The invention provides isolated nucleic acid and amino acid sequences of TL-xcex3, antibodies to TL-xcex3, methods of detecting TL-xcex3 and screening for TL-xcex3 modulators using biologically active TL-xcex3, and kits for screening for TL-xcex3 modulators.
The kinesin superfamily is an extended family of related microtubule motor proteins. This family is exemplified by xe2x80x9ctruexe2x80x9d kinesin, which was first isolated from the axoplasm of squid, where it is believed to play a role in anteograde axonal transport of vesicles and organelles (see, e.g., Goldstein, Annu. Rev. Genet. 27:319-351 (1993)). Kinesin uses ATP to generate force and directional movement associated with microtubules (from the minus to the plus end of the microtubule, hence it is a xe2x80x9cplus-end directedxe2x80x9d motor). Kinesin superfamily members are defined by a kinesin-like motor that is about 340 amino acids in size and shares approximately 35-45% identity (or more) with the xe2x80x9ctruexe2x80x9d kinesin motor domain. Typically, the motor is attached to a variety of tail domains that provide different binding activities to the various kinesin superfamily members.
The kinesin superfamily encompasses a number of families that exhibit a variety of microtubule motor functions, e.g., vesicle and organelle transport, mitotic spindle function, and meiotic spindle function. One such family is the xe2x80x9cunc-104 familyxe2x80x9d named after unc-104 protein in C. elegans (Otsuka et al., Neuron 6:13-122 (1991)). Other members of the unc-104 family include mouse KiflA (murine homolog of unc-104) and KiflB, and human ATSV (homolog of KiflA) (Aizawa et al., J. Cell. Biol., 119:1287-1296 (1992); Okada et al., Cell 81:769-780 (1995); Nangaku et al., Cell 79:1209-1220 (1994); and Furlong et al., Genomics 33:421-429 (1996)). These proteins typically work as monomers, are ATP dependent, and have plus end-directed microtubule motor activity involved in fast anterograde organelle transport in neurons. Fast anterograde transport is a directional transport, typically of membraneous organelles such as mitochondria, other organelles and vesicles such as synaptic vesicles, from the cell body to the top of axon. Members of the unc-104 family are not found in the first completely sequenced genome of S. cerevisae, and on this basis it was believed that fungi did not possess members of the unc-104 motor protein family.
Accordingly, among the objects of this invention is to provide novel members of the kinesin superfamily. It is particularly an object to provide novel kinesins and nucleic acids encoding such kinesins which have anterograde axonal transport activity, including novel members of the unc-104 family, indicated by example, by sequence similarity to known kinesins having such activity. It is also an object to provide methods of use of the compounds provided herein, including methods of diagnosis, treatment of disorders related to the nervous system, treatment of disorders related to fungal infection, bioagricultural, including crop protection, and veterinary applications, and methods of identifying binding agents and modulators of the compounds provided herein.
The present invention provides for the first time TL-xcex3, an ATP-dependent, plus end-directed microtubule motor protein that is a member of the unc-104 family and the kinesin superfamily. Previously, the unc-104 family was not thought to exit in fungi. However, the present invention surprisingly provides identification and cloning of a nucleic acid encoding TL-xcex3 from a hyphal fungus.
In one aspect, the invention provides an isolated nucleic acid sequence encoding a kinesin superfamily, plus end-directed micotubule motor protein, wherein the motor protein has the following properties: (i) the protein""s activity includes plus end-directed microtubule motor activity; and (ii) the protein has a tail domain that has greater than 60% amino acid sequence identity to a TL-xcex3 tail domain as measured using a sequence comparison algorithm.
In one embodiment, the protein further specifically binds to polyclonal antibodies raised against TL-xcex3 of SEQ ID NO:1.
In one embodiment, the nucleic acid encodes TL-xcex3. In another embodiment, the nucleic acid encodes SEQ ID NO:1. In another embodiment, the nucleic acid has a nucleotide sequence of SEQ ID NO:2.
In one embodiment, the sequence comparison algorithm is PILEUP.
In one embodiment, the nucleic acid is amplified by primers that selectively hybridize under stringent hybridization conditions to the same sequence as the primer set:
5xe2x80x2 ATGTCGGGCGGTGGAAATATC 3xe2x80x2 (SEQ ID NO:3) and 5xe2x80x2 GAATTCCTGCTTCGCTGTTTTCA 3xe2x80x2 (SEQ ID NO:4). In another embodiment, the nucleic acid selectively hybridizes under stringent hybridization conditions to SEQ ID NO:2.
In one embodiment, the nucleic acid has identity to a TL-xcex3 derived from a hyphal fungi. In another embodiment, the nucleic acid has identity to the TL-xcex3 derived from Thermomyces lanuginosus. 
In another aspect, the invention provides an explosion vector comprising a nucleic acid encoding a kinesin superfamily, plus end-directed microtubule motor protein, wherein the motor protein has one or more of the properties described above.
In one embodiment, the invention provides a host cell transfected with the vector.
In another aspect, the invention provides an isolated kinesin superfamily, plus end-directed microtubule motor protein, wherein the protein has one or more of the properties described above.
In one embodiment, the protein specifically binds to polyclonal antibodies generated against a tail, motor, or stalk domain of TL-xcex3. In another embodiment, the protein comprises an amino acid sequence of a TL-xcex3 motor domain of SEQ ID NO:1.
In another aspect, the invention provides an antibody that specifically binds to TL-xcex3.
In one embodiment, the antibody specifically binds to a tail, motor, or stalk domain of TL-xcex3.
In another embodiment, the invention further provides chimeric antibodies, humanized antibodies, and the nucleic acids encoding the antibodies provided herein.
In another aspect, the invention provides a method for diagnosing hyphal fungal infections by detecting the presence of TL-xcex3 in a sample, the method comprising the steps of: (i) obtaining a biological sample; (ii) contacting the biological sample with a TL-xcex3 specific reagent that selectively binds to TL-xcex3; and (iii) detecting the level of TL-xcex3 specific reagent that selectively associates with the sample.
In one embodiment, the TL-xcex3 specifies reagent is selected from the group consisting of: TL-xcex3 specific antibodies, TL-xcex3 specific oligonucleotide primers, and TL-xcex3 nucleic acid probes. In another embodiment, the sample is from a plant or vertebrate, preferably a human. In another embodiment, the specific reagent is part of a gene or protein array.
In another aspect, the invention provides a method for screening for modulators of TL-xcex3, the method comprising the steps of: (i) contacting biologically active TL-xcex3 with at least one candidate agent at a test and control concentration and detecting whether a change in TL-xcex3 activity occurs between the test and control concentration, wherein a change indicates a modulator of TL-xcex3. In one embodiment, the activity is selected from the group consisting of plus-end direction microtubule motor activity, ATPase activity and binding activity.
In one embodiment, the method further comprises the step of isolating biologically active TL-xcex3 from a cell sample. In another embodiment, the biologically active TL-xcex3 is recombinant. In another embodiment, the biologically active TL-xcex3 comprises a motor, stalk or tail domain having identity to a motor, stalk or tail domain of Thermomyces lanuginosus TL-xcex3. In another embodiment, the biologically active TL-xcex3 comprises an amino acid sequence of a TL-xcex3 motor domain of SEQ ID NO:1.
In another aspect, the invention provides a kit for screening for modulators of TL-xcex3, the kit comprising: (i) a container holding biologically active TL-xcex3; and (ii) instructions for assaying for TL-xcex3 activity, wherein the TL-xcex3 activity is plus end-directed microtubule motor activity, binding activity, or ATPase activity.
In another aspect, the invention provides, in a computer system, a method of screening for mutations of kinesin superfamily, plus end-directed microtubule motor protein genes, the method comprising the steps of: (i) receiving input of a first nucleic acid sequence encoding a plus end-directed microtubule motor protein having a nucleotide sequence of SEQ ID NO:2 and conservatively modified versions thereof; (ii) comparing the first nucleic acid sequence with a second nucleic acid sequence having substantial identity of the first nucleic acid sequence; and (iii) identifying nucleotide differences between the first and second nucleic acid sequences.
In another aspect, the invention provides, in a computer system, a method for identifying a three-dimensional structure of kinesin superfamily, plus end-directed microtubule motor proteins, the method comprising the steps of: (i) receiving input of an amino acid sequence of at least 10 amino acids of a plus end-directed microtubule motor protein or a nucleotide sequence of at least 30 nucleotides of a gene encoding the motor protein, the protein having an amino acid sequence of SEQ ID NO:1 and conservatively modified versions thereof; and (ii) generating a three-dimensional structure of the protein encoded by the amino acid sequence.
In one aspect, the nucleic acid comprises a sequence which encodes an amino acid sequence which has one or more of the following characteristics:
greater than 60% sequence identity with SEQ ID NO:1, preferably greater than 70%, more preferably greater than 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:1;
greater than 70% sequence identity with amino acids 1-357 of SEQ ID NO:1, more preferably greater than 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with amino acids 1-357 of SEQ ID NO:1;
greater than 60% sequence identity with amino acids 443-601 of SEQ ID NO:1, preferably greater than 70%, more preferably greater than 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with amino acids 443-601 of SEQ ID NO:1;
greater than 50 or 55% sequence identity with amino acids 602-784 of SEQ ID NO:1, preferably greater than 65%, more preferably greater than 70 or 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with amino acids 602-784 of SEQ ID NO:1.
In one embodiment, the nucleic acid comprises a sequence which has one or more of the following characteristics:
greater than 55 or 60% sequence identity with SEQ ID NO:2, preferably greater than 70%, more preferably greater than 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2;
greater than 65% sequence identity with nucleotides 1-1071 of SEQ ID NO:2 more preferably greater than 70 or 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with nucleotides 1-1071 of SEQ ID NO:2;
greater than 55%, preferably greater than 65%, more preferably greater than 70 or 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with nucleotides 1327-1803 or SEQ ID NO:2;
greater than 45 or 55%, preferably greater than 65%, more preferably greater than 70 or 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with nucleotides 1804-2352 of SEQ ID NO:2.
In another embodiment provided herein, the nucleic acid hybridizes under stringent conditions to a nucleic acid having a sequence or complementary sequence thereof selected from the group consisting of SEQ ID NO:2, nucleotides 1-1071 of SEQ ID NO:2, nucleotides 1327-1803 of SEQ ID NO:2, and 1804-2352 of SEQ ID NO:2.
In one aspect, the protein provided herein comprises an amino acid sequence which has one or more of the following characteristics:
greater than 60% sequence identity with SEQ ID NO:1, preferably greater than 70%, more preferably greater than 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:1;
greater than 70% sequence identity with amino acid 1-357 of SEQ ID NO:1, more preferably greater than 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with amino acids 1-357 of SEQ ID NO:1;
greater than 60% sequence identity with amino acids 443-601 of SEQ ID NO:1, preferably greater than 70%, more preferably greater than 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with amino acids 443-601 of SEQ ID NO:1;
greater than 50 or 55% sequence identity with amino acids 602-784 of SEQ ID NO:1, preferably greater than 65%, more preferably greater than 70 or 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with amino acids 602-784 of SEQ ID NO:1.
Not applicable.